Purpose

This page describes the experiment where I investigated gliding speed variations of microtubules using different water isotopes.

This is the third chapter in my completely open notebook science dissertation. If you would like to post questions, comments, or concerns, please join the wiki and post comments to the talk page. If you do not want to join the wiki and would still like to comment, feel free to email me.

The differences for these experiments as compared to others I've done are subtle, however, they make a big difference to the assay. The measurable differences in how the assay responds to different environments is not too surprising considering how sensitive it is to ionic strength and pH (Böhm Stracke & Unger 2000).

New flow cells

The construction of the new flow cells was very similar to the old construction. The only difference was that I intended for the new flow cells to not be permanently sealed. This required finding a material that would "seal" the flow cell in order to prevent evaporation and at the same time allow me to observe it on the microscope, remove it from the microscope, and flow in new material to be observed again. Surprisingly enough, plain old kitchen plastic wrap (cellophane) worked the best at this task.

Step 1: I created a template for fast and reproducible flow cell creation. Using my template, I centered a slide (VWR 48300-025) in the vertical rectangle.

Step 2: I added strips of permanent double stick tape (Scotch) to the slide by using the two most center lines as a guide. This created a channel that is approximately 10 µL in volume. The width of the two center lines was 5 mm.

Step 3: I trimed the excess tape by using the outer lines of the center box as a guide. I did not trim the tape such that the only tape left on the slide was in the center box. I only trimed the tape that hung over the slide. It turns out that keeping this extra tape is useful when making resealable flow cells. After trimming the tape I placed a cover slip (VWR 48366-045) over the center box. The center box was used as a guide to ensure that the cover slip was centered on the slide.

Step 4: I ensured proper adhesion of the cover slip to the tape by pressing it to the slide. Proper adhesion was done when the light that is scattered from the slide + tape + slip combo differs from when the slip is merely placed on the tape. Compare the figure from step 3 to this step and notice the difference in how the light scatters from the center box.

Step 5: If the flow cell was not intended to be used as a resealable one, then it was finished with the previous step. If it was intended to be used with the cellophane sealer, then I continued on with the following steps. Two very thin pieces of double stick tape were placed at the entrances of the flow cell. They have been colored red in the image to enhance contrast. These pieces of tape were essential for proper sealing with a piece of cellophane and helped prevent the objective oil from seeping into the flow cell. They can be made by holding two razor blades together and cutting a thin piece. I had to ensure that the thin pieces of tape were as close as possible to the entrances of the flow cell since I observed excess evaporation occurred if they were not close.

Step 6: Small strips of cellophane (Glad Cling Wrap) were then placed over the flow cells and wrapped around the slide.

I should note that I also tried static cling vinyl and pallet wrap before settling on using cellophane. The static cling vinyl was not used because it was too thick and did not properly seal the flow cell. But, it did indicate to me that the static cling was what I wanted to use as the new sealer for the flow cells. This lead me to pallet wrap which was not used because it contains a small amount of glue and I did not want that glue seeping into my flow cells. Using it did show me that I needed a thinner material to "seal" the flow cell with and ultimately lead to the use of cellophane that can be purchased at a grocery store.

There has been some debate about the use of nail polish as the sealant for flow cells due to the organic solvents that are in them. Using both the nail polish sealant method and this new cellophane seal method (which has no chemicals that could possibly leach into the flow cell) did not show a difference in the observed gliding motility speeds. I will discuss this in greater detail and show a figure below in the Results section.

New solutions

Along with the 10x concentrated solution of PEM mixed in 18.2 MΩ-cm water, I prepared two other solutions containing two different isotopes of water, namely D2O and H218O. Water is often overlooked when investigating molecular systems (Parsegian 1995). The reason for changing the water isotope was to try and measure speed differences dependent on the water isotope concentration in solution. The goal was to try and see if we could somehow relate the change in water isotopes to an increase in osmotic pressure of the system. I will discuss this more in the following sections however, the data is lending us to believe that I measured a viscosity change and not an osmotic pressure change.

D2O overview

D2O has some rather remarkable properties that are completely different than the properties of regular water. For instance, D2O is 25% more viscous than regular water and it has a higher boiling point and a higher melting temperature than regular water (Katz 1957).

D2O has also been shown to stabilize tubulin (Chakrabarti 1999, Das 2008) and is known to prevent microtubules from depolymerizing (Panda 2000). It is used in the thermal stabilization of vaccines (Sen 2009) and can even be used to make fruit flies more tolerant to heat (Pittendrigh 1974). Rhodamine has been shown to have better photostability in heavy hydrogen water as well (Sinha 2002). It is known to prevent cellular growth without changing the permeability of cellular walls to water (Lucké 1935, Lewis 193 & 1934). This last point is very interesting and in order to investigate the effects of D2O on growth, I have started a side project that is attempting to recreate the seminal work done by Lewis. Deuterium is such an odd quantity that there have even been studies showing that a lack of deuterium during growth causes stunting (Somlyai 1993).

Some work has been done on investigating how molecular motors act in a solution of D2O (Chaen 2001). Chaen et. al. also showed that when they reduced the amount of ATP in solution such that it became the rate limiting step, the speed at which actomyosin converted ATP was less than the speed in H2O.

H-PEM recipe

No studies have been made on how water isotopes affect kinesin and microtubules. To remedy this, I took data looking at the speed at which microtubules glide at in the presence of D2O. In order to do this, I prepared a solution of PEM in D2O which I called H-PEM to indicate that it contained the heavy hydrogen isotope of water. H-PEM was made in a 10x concentrated solution and contained the following.

I used NaOH to pH the solution but, pH is a concentration measurement of hydrogen ions in solution and not deuterium ions. In order to measure the correct "pD" of this solution using the pH meter, I had to add 0.41 to the measured value (Covington 1968). This necessitated that the solution of PEM in D2O was pH-ed to 7.30 using an approximate amount of NaOH. I designated this solution as H-PEM to indicate it was the heavy hydrogen water buffer. It was passed through a 0.2 μm syringe filter, aliquoted, and then stored at 4˚C in screw top vials.

H218O overview

Heavy oxygen water is 5% more viscous than regular water (Kudish 1972). There is not much in the literature about heavy oxygen water other than it is used extensively in labeling (Schwartz 2007, Dawis 1989). Its diffusion through regular H216O water has been measured as well (Easteal 1984).

The lack of research using H218O can probably be attributed to its high cost. A single gram at 97% 18O purity costs $200. A gram with 99% atomic purity of 18O costs nearly $1000 at the time of this writing. With this type of cost prohibitions, I had to change the way I prepared H218O water with PEM in it.

O-PEM recipe

To prepare the PEM solution using heavy oxygen water I actually diluted the 10x PEM into the H218O water. This allowed me to make a solution of PEM containing 90% H218O. To make this solution I did the following.

100 μL of 10x PEM

900 μL of H218O

I should note that the vial in which H218O comes in makes it extremely difficult to remove all the material. Because of this, I was only able on average to acquire 850 μL of H218O water from a single vial. Using the techniques outlined in the previous chapters, I was able to run 30 different assays using varying amounts of H218O water in the assay with only 2 stock vials of heavy oxygen water. This solution was not pH-ed and it was not filtered for fear of loosing too much material in the filter. It aliquoted it and stored it at 3°C.

D-PEM recipe

Experiment and data analysis

The experimental procedure and data analysis is nearly identical to the steps taken in Chapter 2. Below, I will outline the experimental procedure since it does differ from what was done in Chapter 2 and is relevant to the discussion below. However, I will only link to the data analysis section of Chapter 2 below.

Experiment

Experimental flow cells for observation were prepared as follows.

Prepared a flow cell. Either the type to be sealed with nail polish or the type to be sealed with cellophane.

During the 10 minute incubation, kinesin was diluted in 0.5 mg/mL α-casein in PEM plus 1 mM ATP. The final concentration of kinesin used in these experiments was 27.5 μg/mL.

After 10 minutes, kinesin was flowed into the flow cell by fluid exchange. The volume of the flow cell was approximately 10 μL. To ensure that all the fluid in the flow cell was exchanged properly, 20 μL of the kinesin mixture was flowed into the chamber. This was then allowed to sit for 5 minutes.

During the various incubation steps, a new motility solution was prepared for each assay. The motility assays consisted of:

After the 5 minute incubation with kinesin, 20 μL of the motility solution was introduced to the flow cell by fluid exchange.

The flow cell was either sealed with nail polish or cellophane dependent on which assay was to be performed.

After sealing the flow cell, the slide was immediately placed on the microscope for observation. For a detailed description of why I used these parameters, please see Chapter 2. Briefly, the camera was set take images as follows.

Data analysis

Results and discussion

Results

Figure X: Graph showing the gliding speed dependence of microtubules when D2O is added to solution.

Each slide was observed for a total of 15 fields of view, or about 30 minutes for each slide. Assays were run at 10% increments of H-PEM added to the motility solution from 0 - 90%. Each 10% increment was then run a total of 3 times. For each slide, only the last 10 fields of view were kept to compute an average speed value for that assay. The first 5 data points were removed due to the increase in temperature of the slide from room temperature (24°C) to the objective at 33.1°C. This gave a total of 3 average velocity data points that are the accumulation of hundreds of tracked microtubules.

Each data point in Figure X is the average of those 3 speed measurements and the error bars are the standard error of the mean (SEM). The concentration of D2O was calculated by first converting the percentage values to concentration values in one mole of H2O. Next, the baseline amount of D2O in water was calculated to be 8 mM (Somlyai 1993). The 8 mM baseline was then added to the concentration of H-PEM used.